Bone marrow mesenchymal stromal cells support regeneration of intestinal damage in a colitis mouse model, independent of their CXCR4 expression.

Inflammatory bowel disease (IBD) is characterized by a chronically dysregulated immune response in the gastrointestinal tract. Bone marrow multipotent mesenchymal stromal cells have an important immunomodulatory function and support regeneration of inflamed tissue by secretion of soluble factors as well as through direct local differentiation. CXCR4 is the receptor for CXCL12 (SDF-1, stromal-derived factor-1) and has been shown to be the main chemokine receptor, required for homing of MSCs. Increased expression of CXCL12 by inflamed intestinal tissue causes constitutive inflammation by attracting lymphocytes but can also be used to direct MSCs to sites of injury/inflammation. Trypsin is typically used to dissociate MSCs into single-cell suspensions but has also been shown to digest surface CXCR4. Here, we assessed the regenerative effects of CXCR4high and CXCR4low MSCs in an immune-deficient mouse model of DSS-induced colitis. We found that transplantation of MSCs resulted in clinical improvement and histological recovery of intestinal epithelium. In contrary to our expectations, the levels of CXCR4 on transplanted MSCs did not affect their regenerative supporting potential, indicating that paracrine effects of MSCs may be largely responsible for their regenerative/protective effects.

Allogeneic bone marrow mesenchymal stem cell-derived exosomes alleviate human hypoxic AKI-on-a-Chip within a tight treatment window.

BACKGROUND: Acute hypoxic proximal tubule (PT) injury and subsequent maladaptive repair present high mortality and increased risk of acute kidney injury (AKI) - chronic kidney disease (CKD) transition. Human bone marrow mesenchymal stem cell-derived exosomes (hBMMSC-Exos) as potential cell therapeutics can be translated into clinics if drawbacks on safety and efficacy are clarified. Here, we determined the real-time effective dose and treatment window of allogeneic hBMMSC-Exos, evaluated their performance on the structural and functional integrity of 3D microfluidic acute hypoxic PT injury platform. METHODS: hBMMSC-Exos were isolated and characterized. Real-time impedance-based cell proliferation analysis (RTCA) determined the effective dose and treatment window for acute hypoxic PT injury. A 2-lane 3D gravity-driven microfluidic platform was set to mimic PT in vitro. ZO-1, acetylated α-tubulin immunolabelling, and permeability index assessed structural; cell proliferation by WST-1 measured functional integrity of PT. RESULTS: hBMMSC-Exos induced PT proliferation with ED50 of 172,582 µg/ml at the 26th hour. Hypoxia significantly decreased ZO-1, increased permeability index, and decreased cell proliferation rate on 24-48 h in the microfluidic platform. hBMMSC-Exos reinforced polarity by a 1.72-fold increase in ZO-1, restored permeability by 20/45-fold against 20/155 kDa dextran and increased epithelial proliferation 3-fold compared to control. CONCLUSIONS: The real-time potency assay and 3D gravity-driven microfluidic acute hypoxic PT injury platform precisely demonstrated the therapeutic performance window of allogeneic hBMMSC-Exos on ischemic AKI based on structural and functional cellular data. The novel standardized, non-invasive two-step system validates the cell-based personalized theragnostic tool in a real-time physiological microenvironment prior to safe and efficient clinical usage in nephrology.

Dual Strategy with Adipose-Derived Stem Cells and l-arginine Recovered Cavernosal Functions in a Rat Model of Radical Prostatectomy.

As standard therapy for prostate cancer, radical prostatectomy causes cavernous nerve (CN) injury and increases fibrosis and hypoxia-induced penile structural alterations. This study aimed to determine the potential beneficial effects of adipose-derived stem cells (ADSCs) and l-arginine alone or in combination on the penile erection in a rat model of erectile dysfunction caused by bilateral cavernous nerve transection (CNT). Male rats (n = 35) were randomized into five groups: Sham-operated; CNT (4-weeks); CNT plus ADSCs (1 × 106 cells by intracavernosal injection); CNT plus l-arginine (4 weeks, 10 mg/kg/day, oral); and ADSCs combined with l-arginine in CNT. In vivo erectile responses and in vitro relaxant responses were measured. Western blot and immunohistochemistry analyses were used to determine the expression and localization of endothelial nitric oxide synthase, neuronal nitric oxide synthase, transforming growth factor-beta 1, hypoxia-inducible factor-1 alpha (HIF-1α), and apoptosis markers (Bax and Bcl-2). The ratio of smooth muscle to collagen and nerve regeneration were calculated using Masson's trichrome and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining. The combined treatment restored diminished erectile responses, endothelium-dependent acetylcholine, and electrical field stimulation-induced relaxation of the corpus cavernosum in rats with CNT, whereas either monotherapy produced only partial improvements. All treatment regimens restored increases in the protein expression of HIF-1 and Bax in rats with CNT. The decrease in smooth muscle mass and NADPH-diaphorase-positive nerve fibers was partially ameliorated by monotherapy, whereas combined therapy led to recovery. These findings indicate that combined treatment with ADSCs and l-arginine may restore erectile function in rats with CNT by inhibiting hypoxia-induced neurotoxicity and preserving endothelium function and smooth muscle content.

A pilot study: Nano-hydroxyapatite-PEG/PLA containing low dose rhBMP2 stimulates proliferation and osteogenic differentiation of human bone marrow derived mesenchymal stem cells.

Background: Bone morphogenetic protein 2 (BMP2) can enhance posterolateral spinal fusion (PLSF). The minimum effective dose that may stimulate mesenchymal stem cells however remains unknown. Nano-hydroxyapatite (nHAp) polyethylene glycol (PEG)/polylactic acid (PLA) was combined with recombinant human BMP2 (rhBMP2). We in vitro evaluated proliferation, differentiation, and osteogenic genes of human bone marrow mesenchymal stem cells with 0.5, 1.0, and 3.0 µg/mL rhBMP2 doses in this study. Methods: In vitro experimental study was designed to proliferation by a real-time quantitative cell analysis system and the osteogenic differentiation by alkaline phosphatase (ALP) activity and osteogenic marker (Runx2, OPN, and OCN) gene expressions of human derived bone marrow mesenchymal stem cells (hBMMSCs). nHAp was produced by wet chemical process and characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, and energy-dispersive x-ray spectroscopy. PEG/PLA polymer was produced at a 51:49 molar ratio. 0.5, 1.0, and 3.0 µg/mL rhBMP2 and nHAp was combined with the polymers. hBMMSCs were characterized by multipotency assays and surface markers were assessed by flow cytometer. The hBMMSC-rhBMP2 containing nHAp-PEG/PLA composite interaction was evaluated by transmission electron microscopy. Proliferative effect was evaluated by real-time proliferation analysis, and osteogenic capacity was evaluated by ALP activity assay and qPCR. Results: hBMMSC proliferation in the 0.5 µg/mL rhBMP2 + nHAp-PEG/PLA and the 1.0 µg/mL rhBMP2 + nHAp-PEG/PLA groups were higher compared to control. 1.0 µg/mL rhBMP2 + nHAp-PEG/PLA and 3.0 µg/mL rhBMP2 + nHAp-PEG/PLA containing composites induced ALP activity on days 3 and 10. 0.5 µg/mL rhBMP2 + nHAp-PEG/PLA application stimulated Runx2 and OPN gene expressions. Conclusion: rhBMP2 + nHAp-PEG/PLA composites stimulate hBMMSC proliferation and differentiation. The nHAp-PEG/PLA composite with low dose of rhBMP2 may enhance bone formation in future clinical PLSF applications.

CB65 and novel CB65 liposomal system suppress MG63 and Saos-2 osteosarcoma cell growth in vitro.

Curable approaches for primary osteosarcoma are inadequate and urge investigation of novel therapeutic formulations. Cannabinoid ligands exert antiproliferative and apoptotic effect on osteosarcoma cells via cannabinoid 2 (CB2) or transient receptor potential vanilloid type (TRPV1) receptors. In this study, we confirmed CB2 receptor expression in MG63 and Saos-2 osteosarcoma cells by qRT-PCR and flow cytometry (FCM), then reported the reduction effect of synthetic specific CB2 receptor agonist CB65 on the proliferation of osteosarcoma cells by WST-1 (water-soluble tetrazolium-1) and RTCA (real-time impedance-based proliferation). CB65 revealed an IC50 (inhibitory concentration) for MG63 and Saos-2 cells as 1.11 × 10-11 and 4.95 × 10-11 M, respectively. The specific antiproliferative effect of CB65 on osteosarcoma cells was inhibited by CB2 antagonist AM630. CB65 induced late apoptosis of MG63 and Saos-2 cells at 24 and 48 h, respectively by FCM when applied submaximal concentration. A novel CB65 liposomal system was generated by a thin film hydration method with optimal particle size (141.7 ± 0.6 nm), polydispersity index (0.451 ± 0.026), and zeta potential (-10.9 ± 0.3 mV) values. The encapsulation efficiency (EE%) of the CB65-loaded liposomal formulation was 51.12%. The CB65 and CB65-loaded liposomal formulation releasing IC50 of CB65 reduced proliferation by RTCA and invasion by scratch assay and induced late apoptosis of MG63 and Saos-2 cells, by FCM. Our results demonstrate the CB2 receptor-mediated antiproliferative and apoptotic effect of a new liposomal CB65 delivery system on osteosarcoma cells that can be used as a targeted and intelligent tool for bone tumors to ameliorate pediatric bone cancers following in vivo validation.

A pumpless monolayer microfluidic device based on mesenchymal stem cell-conditioned medium promotes neonatal mouse in vitro spermatogenesis.

BACKGROUND: Childhood cancer treatment-induced gonadotoxicity causes permanent infertility/sub-infertility in nearly half of males. The current clinical and experimental approaches are limited to cryopreservation of prepubertal testicular strips and in vitro spermatogenesis which are inadequate to achieve the expanded spermatogonial stem/progenitor cells and spermatogenesis in vitro. Recently, we reported the supportive effect of bone marrow-derived mesenchymal cell co-culture which is inadequate after 14 days of culture in static conditions in prepubertal mouse testis due to lack of microvascular flow and diffusion. Therefore, we generated a novel, pumpless, single polydimethylsiloxane-layered testis-on-chip platform providing a continuous and stabilized microfluidic flow and real-time cellular paracrine contribution of allogeneic bone marrow-derived mesenchymal stem cells. METHODS: We aimed to evaluate the efficacy of this new setup in terms of self-renewal of stem/progenitor cells, spermatogenesis and structural and functional maturation of seminiferous tubules in vitro by measuring the number of undifferentiated and differentiating spermatogonia, spermatocytes, spermatids and tubular growth by histochemical, immunohistochemical, flow cytometric and chromatographic techniques. RESULTS: Bone marrow-derived mesenchymal stem cell-based testis-on-chip platform supported the maintenance of SALL4(+) and PLZF(+) spermatogonial stem/progenitor cells, for 42 days. The new setup improved in vitro spermatogenesis in terms of c-Kit(+) differentiating spermatogonia, VASA(+) total germ cells, the meiotic cells including spermatocytes and spermatids and testicular maturation by increasing testosterone concentration and improved tubular growth for 42 days in comparison with hanging drop and non-mesenchymal stem cell control. CONCLUSIONS: Future fertility preservation for male pediatric cancer survivors depends on the protection/expansion of spermatogonial stem/progenitor cell pool and induction of in vitro spermatogenesis. Our findings demonstrate that a novel bone marrow-derived mesenchymal stem cell-based microfluidic testis-on-chip device supporting the maintenance of stem cells and spermatogenesis in prepubertal mice in vitro. This new, cell therapy-based microfluidic platform may contribute to a safe, precision-based cell and tissue banking protocols for prepubertal fertility restoration in future.

Synergistic Antitumor Potency of a Self-Assembling Cyclodextrin Nanoplex for the Co-Delivery of 5-Fluorouracil and Interleukin-2 in the Treatment of Colorectal Cancer.

Chemotherapy is the most used method after surgery in the treatment of colon cancer. Cancer cells escape the recognition mechanism of immune system cells to survive and develop chemoresistance. Therefore, the use of immunotherapy in combination with chemotherapy can increase the effectiveness of the treatment. Nanoparticles have been used clinically to increase the accumulation of therapeutics in target tissues and reduce toxicity. In this paper, nanoplexes were formed via cationic cyclodextrin polymer, 5-Fluorouracil, and Interleukin-2 based on the opposite charge interaction of macromolecules without undergoing any structural changes or losing the biological activity of Interleukin-2. Anticancer activities of nanoplexes were determined in two-dimensional and three-dimensional cell culture setups. The dual drug-loaded cyclodextrin nanoplexes diffused deeper into the spheroids and accelerated apoptosis when compared with 5-FU solutions. In the colorectal tumor-bearing animal model, survival rate, antitumor activity, metastasis, and immune response parameters were assessed using a cyclodextrin derivative, which was found to be safe based on the ALT/AST levels in healthy mice. Histomorphometric analysis showed that the groups treated with the nanoplex formulation had significantly fewer initial tumors and lung foci when compared with the control. The dual drug-loaded nanoplex could be a promising drug delivery technique in the immunochemotherapy of colorectal cancer.

Comparison of total anastomosis time between four different combinations of suturing and knot tying techniques in microsurgical anastomosis.

BACKGROUND: Various techniques have been described for performing microsurgical anastomosis with providing high patency rates. Although the total anastomotic time may not be an issue when dealing with a single set of anastomoses, using a faster technique may save significant amount of time in cases of transferring flaps with shorter critical ischemia time or where multiple anastomoses are required. This study compares the total anastomosis time between four different combinations of commonly used suturing and knot tying techniques. METHODS: Twenty-four rats were divided into 4 groups. Simple interrupted suture with conventional knot tying technique (SIS-CT) was used in group I, continuous suture technique with conventional knot tying (CST) was used in group II, simple interrupted suture with airborne knot tying technique(SIS-AT) was used in group III, and continuous-interrupted suture with airborne knot tying technique(CIS-AT) was used in group IV for microsurgical anastomosis. Total anastomosis time and patency rates with each technique and samples from anastomotic sites were analyzed. RESULTS: The mean time required for microvascular anastomosis of the femoral artery was 1075 s in group I, 799 s in group II, 844 s in group III, and 973 s in group IV. The difference between four groups was statistically significant. The anastomoses in group II and group III were completed in the shortest period of time. Intergroup comparison revealed that the difference between group II and group III was not statistically significant, however, total anastomosis time for completion of the anastomosis was significantly longer for group I, followed by group IV. Thrombosis rates and histological analysis revealed no significant differences among four groups. CONCLUSION: CST and SIS-AT techniques can significantly reduce microsurgical anastomosis time and provide high patency rates. Also, the time needed to complete an anastomosis was significantly shorter for CIS-AT when compared to SIS-CT.

Topical Intranasal Insulin Enhances Healing of Nasal Mucosa: An Experimental Animal Study.

OBJECTIVE: Aim of this study was to evaluate the effect of topical intranasal insulin on healing of nasal mucosa in a rat model. METHODS: Forty-eight Wistar rats, weighing between 250 and 300 g and aged 10-12 weeks were used and randomized into two equal groups. 1.9 mm curette was introduced through the left nostril and 1.9 mm mucosa from the left nasal septum was curetted. Postoperatively, animals in the control group received 1 mL of physiologic saline, 3 times a day in a nasal irrigation fashion. Animals in the experimental group received 1 mL of 5 IU/mL regular insulin in saline solution. Subjects were sacrificed after 5, 10, and 15 days and macroscopic and histomorphometric evaluations were performed. RESULTS: There were no mucosal synechiae and septal perforation macroscopically. Histological examination revealed that the defect size reduction was 21% in the saline group versus 56% in the insulin group on the fifth day (p = 0.006). There was 62% defect reduction in the saline group versus 79% in the insulin group on the 10th day (p = 0.034). On the 15th day, only 67% of saline group animals had complete defect closure, whereas 100% of animals treated with insulin had complete closure (92% vs 100% mucosal defect reduction, p = 0.036). Both edema and inflammation were less in the insulin group on 15th day (p = 0.006; p = 0.023, respectively). CONCLUSION: The results from this study support the safety and efficacy of topical insulin on wound healing in the literature. This study could guide further experimental studies that examine human sinonasal wound healing.

Cannabinoids as Prospective Anti-Cancer Drugs: Mechanism of Action in Healthy and Cancer Cells.

Endogenous and exogenous cannabinoids modulate many physiological and pathological processes by binding classical cannabinoid receptors 1 (CB1) or 2 (CB2) or non-cannabinoid receptors. Cannabinoids are known to exert antiproliferative, apoptotic, anti-migratory and anti-invasive effect on cancer cells by inducing or inhibiting various signaling cascades. In this chapter, we specifically emphasize the latest research works about the alterations in endocannabinoid system (ECS) components in malignancies and cancer cell proliferation, migration, invasion, angiogenesis, autophagy, and death by cannabinoid administration, emphasizing their mechanism of action, and give a future perspective for clinical use.

Thioredoxin System and miR-21, miR-23a/b and let-7a as Potential Biomarkers for Brain Tumor Progression: Preliminary Case Data.

BACKGROUND: The thioredoxin system and microRNAs (miRNAs) are potential targets for both cancer progression and treatment. However, the role of miRNAs and their relation with the expression profile of thioredoxin system in brain tumor progression remains unclear. METHODS: In this study, we aimed to determine the expression profiles of redox components Trx-1, TrxR-1 and PRDX-1, and oncogenic miR-21, miR-23a/b and let-7a and oncosuppressor miR-125 in different brain tumor tissues and their association with increasing tumor grade. We studied Trx-1, TrxR-1, and PRDX-1 messenger RNA expression levels by quantitative real-time polymerase chain reaction and protein levels by Western blot and miR-23a, miR-23b, miR-125a, miR-21, and let-7a miRNA expression levels by quantitative real-time polymerase chain reaction in 16 glioma, 15 meningioma, 5 metastatic, and 2 benign tumor samples. We also examined Trx-1, TrxR-1, and PRDX-1 protein levels in serum samples of 36 patients with brain tumor and 37 healthy volunteers by enzyme-linked immunosorbent assay. RESULTS: We found that Trx-1, TrxR-1, and PRDX-1 presented high messenger RNA expression but low protein expression in low-grade brain tumor tissues, whereas they showed higher protein expression in sera of patients with low-grade brain tumors. miR-23b, miR-21, miR-23a, and let-7a were highly expressed in low-grade brain tumor tissues and positively correlated with the increase in thioredoxin system activity. CONCLUSIONS: Our findings showed that Trx-1, TrxR-1, miR-21, miR-23a/b, and let-7a might be used for brain tumor diagnosis in the clinic. Further prospective studies including molecular pathway analyses are required to validate the miRNA/Trx system regulatory axis in brain tumor progression.

Mesenchymal stem cells promote spermatogonial stem/progenitor cell pool and spermatogenesis in neonatal mice in vitro.

Prepubertal cancer treatment leads to irreversible infertility in half of the male patients. Current in vitro spermatogenesis protocols and cryopreservation techniques are inadequate to expand spermatogonial stem/progenitor cells (SSPC) from testicles. Bone marrow derived mesenchymal stem cells (BM-MSC) bearing a close resemblance to Sertoli cells, improved spermatogenesis in animal models. We asked if a co-culture setup supported by syngeneic BM-MSC that contributes to the air-liquid interphase (ALI) could lead to survival, expansion and differentiation of SSPCs in vitro. We generated an ALI platform able to provide a real-time cellular paracrine contribution consisting of syngeneic BM-MSCs to neonatal C57BL/6 mice testes. We aimed to evaluate the efficacy of this culture system on SSPC pool expansion and spermatogenesis throughout a complete spermatogenic cycle by measuring the number of total germ cells (GC), the undifferentiated and differentiating spermatogonia, the spermatocytes and the spermatids. Furthermore, we evaluated the testicular cell cycle phases, the tubular and luminal areas using histochemical, immunohistochemical and flow cytometric techniques. Cultures in present of BM-MSCs displayed survival of ID4(+) spermatogonial stem cells (SSC), expansion of SALL4(+) and OCT4(+) SSPCs, VASA(+) total GCs and Ki67(+) proliferative cells at 42 days and an increased number of SCP3(+) spermatocytes and Acrosin(+) spermatids at 28 days. BM-MSCs increased the percentage of mitotic cells within the G2-M phase of the total testicular cell cycle increased for 7 days, preserved the cell viability for 42 days and induced testicular maturation by enlargement of the tubular and luminal area for 42 days in comparison to the control. The percentage of PLZF(+) SSPCs increased within the first 28 days of culture, after which the pool started to get smaller while the number of spermatocytes and spermatids increased simultaneously. Our findings established the efficacy of syngeneic BM-MSCs on the survival and expansion of the SSPC pool and differentiation of spermatogonia to round spermatids during in vitro culture of prepubertal mice testes for 42 days. This method may be helpful in providing alternative cures for male fertility by supporting in vitro differentiated spermatids that can be used for round spermatid injection (ROSI) to female oocyte in animal models. These findings can be further exploited for personalized cellular therapy strategies to cure male infertility of prepubertal cancer survivors in clinics.

Vancomycin Containing PDLLA and PLGA/ß-TCP Inhibit Biofilm Formation but Do Not Stimulate Osteogenic Transformation of Human Mesenchymal Stem Cells.

Aims: Chronic osteomyelitis, including implant-related prosthetic joint infection, is extremely difficult to cure. We develop vancomycin containing release systems from poly(d,l-lactide) (PDLLA) and poly(d,l-lactide-co-glycolide) (PLGA) composites with beta-tricalcium phosphate (ß-TCP) to treat methicillin-resistant Staphylococcus aureus osteomyelitis. We ask whether vancomycin containing PDLLA/ß-TCP and PLGA/ß-TCP composites will prevent early biofilm formation, allow cell proliferation and osteogenic differentiation, and stimulate osteogenic signaling molecules in the absence of an osteogenic medium. Methods: Composites were produced and characterized with scanning electron microscopy. In vitro vancomycin release was assessed for 6 weeks. Biofilm prevention was calculated by crystal violet staining. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and osteosarcoma cell (SaOS-2) proliferation and differentiation were assessed with water soluble tetrazolium salt and alkaline phosphatase (ALP) staining. Real-time quantitative polymerase chain reaction defined osteogenic signaling molecules for hBM-MSCs. Results: Totally, 3.1 ± 0.2 mg and 3.4 ± 0.4 mg vancomycin released from PDLLA/ß-TCP and the PLGA/ß-TCP composites, respectively, and inhibited early biofilm formation. hBM-MSCs and SaOS-2 cells proliferated on the composites and stimulated ALP activity of cells. Runt-related transcription factor 2 (RUNX2) and SRY-Box transcription Factor 9 (SOX9) expressions were, however, lower with composites when compared with control. Conclusion: Vancomycin containing PDLLA/ß-TCP and PLGA/ß-TCP composites inhibited early biofilm formation and proliferated and differentiated hBM-MSCs and SaOS-2 cells, but osteogenesis-related RUNX2 and SOX9 transcription factors were not strongly expressed in the absence of an osteogenic medium for 14 days.

Effect of mesenchymal stem cells therapy in experimental kaolin induced syringomyelia model.

BACKGROUND: Syringomyelia is a pathological cavitation of the spinal cord. In this study, we examined whether a syrinx cavity would limit itself with axonal regeneration and stem cell activity in the cavity, and we evaluated subjects on a functional basis. METHODS: Groups were designated as kaolin, trauma, kaolin-trauma, and saline groups. Also divided out of the syringomyelia treated groups were those given human mesenchymal stem cells (hMSCs). All groups were evaluated with immunohistochemistry, electron microscopy, confocal microscopy and functionally. RESULTS: The kaolin-trauma group had a significant correction of BBB score with hMSCs therapy. The syrinx cavity measurements showed significant improvement in groups treated with hMSCs. The tissue surrounding the syrinx cavity, however, appeared to be better organized in groups treated with hMSCs. The process of repair and regeneration of damaged axons in the lesion were more improved in groups treated with hMSCs. Using confocal microscopy, fluorescence of hMSCs was observed in the central canal, in the ependymal tissue, and around the lesion. CONCLUSIONS: It was concluded that axonal repair accelerated in groups receiving stem cells, and thus, stem cells may be effective in recovery of neural tissue and myelin damage in syringomyelia.

Human laryngeal squamous cell carcinoma cell line release of endogenous anandamide and 2-arachidonoylglycerol, and their antiproliferative effect via exogenous supplementation: an in vitro study.

The level of the major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are altered in several types of carcinomas, and are known to regulate tumor growth. Thusly, this study hypothesized that the HEp-2 human laryngeal squamous cell carcinoma (LSCC) cell line releases AEA and 2-AG, and aimed to determine if their exogenous supplementation has an anti-proliferative effect in vitro. In this in vitro observational study a commercial human LSCC cell line (HEp-2) was used to test for endogenous AEA and 2-AG release via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The anti-proliferative effect of AEA and 2-AG supplementation was evaluated via WST-1 proliferation assay. It was observed that the HEp-2 LSCC cell line released AEA and 2-AG; the median quantity of AEA released was 15.69 ng mL-1 (range: 14.55-15.95 ng mL-1) and the median quantity of 2-AG released was 2.72 ng -1 (range: 2.67-2.74 ng mL-1). Additionally, both AEA and 2-AG exhibited an anti-proliferative effect. The anti-proliferative effect of 2-AG was stronger than that of AEA. These findings suggest that AEA might function via a CB1 receptor-independent pathway and that 2-AG might function via a CB2-dependent pathway. The present findings show that the HEp-2 LSCC cell line releases the major endocannabinoids AEA and 2-AG, and that their supplementation inhibits tumor cell proliferation in vitro. Thus, cannabinoid ligands might represent novel drug candidates for laryngeal cancers, although functional in vivo studies are required in order to validate their potency.

Effects of 5-fluorouracil released from different prosthetic meshes on post-operative adhesion formation in rats.

OBJECTIVE: Post-operative adhesion is a common problem in abdominal surgery. Especially, foreign materials are strong stimulus for the development of adhesions. The aim of this study was to investigate whether drug release material coated prosthetic mesh decreases intra-abdominal adhesion formation or not. METHODS: 5-Fluorouracil (5-FU) releasing "chitosan gels" were loaded to polypropylene and polyglactin-910 grafts. Polypropylene, polyglactin-910 grafts, chitosan gel, and 5-FU-loaded polyglactin 910, polypropylene grafts were used to cover abdominal defects of rats which were created under sterile conditions (n=84). Each group was divided into two subgroups (n=6). Subgroups were sacrificed on the 7th and 30th days. RESULTS: The 7th day macroscopic examinations were similar. Polypropylene group was most adhesive group on the 30th day. There were less adhesions in chitosan gel and 5-FU-loaded groups. Capsule and capsule margins showed no difference on both the 7th and 30th days. Polypropylene-5-FU group and polypropylene-chitosan gel group showed significantly less macroscopic adhesions than polypropylene control group. Furthermore, polyglactin-910-chitosan gel group was less adhesive than polypropylene control group. CONCLUSION: This study showed that 5-FU decreases the adhesions but the dosage and release kinetics need further investigations.

Therapeutic efficacy and gastrointestinal biodistribution of polycationic nanoparticles for oral camptothecin delivery in early and late-stage colorectal tumor-bearing animal model.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world and is the second leading cause of cancer related deaths. New cases are increasingly diagnosed every day, but current therapeutic options are still insufficient for an effective treatment. In CRC treatment, there is a significant need for alternative treatment approaches that can both prevent relapse and provide strong antimetastatic effects as the intestines and colon are prone to metastasis to neighboring organs and tissues as well as the liver and the lung. In this study, optimized polycationic cyclodextrin (CD) nanoparticles for oral Camptothecin (CPT) delivery were comprehensively examined for in vivo performance in early and late stage tumor bearing mouse model in terms of antitumoral and antimetastatic efficacy of CPT bound to polycationic CD nanoparticles in comparison to free CPT. In addition, the gastrointestinal localization of a single administration of fluorescent dye loaded polycationic CD nanoparticles in the gastrointestinal tract at the end of 24 h after oral administration was also imaged and evaluated by in vivo imaging system against fluorescent dye intensity. Results showed that survival percentage was significantly improved in CRC-bearing mice compared to oral CPT solution, with significantly reduced colorectal tumor masses and number of liver metastatic foci (p < 0.05). It was also possible to differentiate between the effectiveness of nanoparticles in early or late stages of CRC. In vivo imaging studies have also confirmed that polycationic CD nanoparticles are able to deliver the therapeutic load up to the colon and tend to accumulate especially in tumor foci, indicating an effective local treatment strategy. In addition number of liver metastases were significantly decreased with the CPT-loaded polycationic CD nanoparticle formulation in both early and late stage tumor models. These findings indicated that CPT-loaded polycationic CD nanoparticles could be an efficient oral nanocarrier formulation for anticancer molecules that have limited application because of oral bioavailability and stability problems.

Stem Cell Applications in Lysosomal Storage Disorders: Progress and Ongoing Challenges.

Lysosomal storage disorders (LSDs) are rare inborn errors of metabolism caused by defects in lysosomal function. These diseases are characterized by accumulation of completely or partially degraded substrates in the lysosomes leading to cellular dysfunction of the affected cells. Currently, enzyme replacement therapies (ERTs), treatments directed at substrate reduction (SRT), and hematopoietic stem cell (HSC) transplantation are the only treatment options for LSDs, and the effects of these treatments depend strongly on the type of LSD and the time of initiation of treatment. However, some of the LSDs still lack a durable and curative treatment. Therefore, a variety of novel treatments for LSD patients has been developed in the past few years. However, despite significant progress, the efficacy of some of these treatments remains limited because these therapies are often initiated after irreversible organ damage has occurred.Here, we provide an overview of the known effects of LSDs on stem cell function, as well as a synopsis of available stem cell-based cell and gene therapies that have been/are being developed for the treatment of LSDs. We discuss the advantages and disadvantages of use of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and induced pluripotent stem cell (iPSC)-related (gene) therapies. An overview of current research data indicates that when stem cell and/or gene therapy applications are used in combination with existing therapies such as ERT, SRT, and chaperone therapies, promising results can be achieved, showing that these treatments may result in alleviation of existing symptoms and/or prevention of progression of the disease. All together, these studies offer some insight in LSD stem cell biology and provide a hopeful perspective for the use of stem cells. Further development and improvement of these stem cell (gene) combination therapies may greatly improve the current treatment options and outcomes of patients with a LSD.

Development, characterization, and hematopoietic differentiation of Griscelli syndrome type 2 induced pluripotent stem cells.

BACKGROUND: Griscelli syndrome type 2 (GS-2) is a rare, autosomal recessive immune deficiency syndrome caused by a mutation in the RAB27A gene, which results in the absence of a protein involved in vesicle trafficking and consequent loss of function of in particular cytotoxic T and NK cells. Induced pluripotent stem cells (iPSC) express genes associated with pluripotency, have the capacity for infinite expansion, and can differentiate into cells from all three germ layers. They can be induced using integrative or non-integrative systems for transfer of the Oct4, Sox2, Klf4, and cMyc (OSKM) transcription factors. To better understand the pathophysiology of GS-2 and to test novel treatment options, there is a need for an in vitro model of GS-2. METHODS: Here, we generated iPSCs from 3 different GS-2 patients using lentiviral vectors. The iPSCs were characterized using flow cytometry and RT-PCR and tested for the expression of pluripotency markers. In vivo differentiation to cells from all three germlines was tested using a teratoma assay. In vitro differentiation of GS-2 iPSCs into hematopoietic stem and progenitor cells was done using Op9 feeder layers and specified media. RESULTS: All GS-2 iPSC clones displayed a normal karyotype (46XX or 46XY) and were shown to express the same RAB27A gene mutation that was present in the original somatic donor cells. GS-2 iPSCs expressed SSEA1, SSEA4, TRA-1-60, TRA-1-81, and OCT4 proteins, and SOX2, NANOG, and OCT4 expression were confirmed by RT-PCR. Differentiation capacity into cells from all three germ layers was confirmed using the teratoma assay. GS-2 iPSCs showed the capacity to differentiate into cells of the hematopoietic lineage. CONCLUSIONS: Using the lentiviral transfer of OSKM, we were able to generate different iPSC clones from 3 GS-2 patients. These cells can be used in future studies for the development of novel treatment options and to study the pathophysiology of GS-2 disease.

Chitosan Channels Stuffed with Mesenchyme Originated Stem/Progenitor Cells for Renovate Axonal Regeneration in Complete Spinal Cord Transection.

AIM: To examine the implantation of chitosan channels stuffed with mesenchyme-originated stem/progenitor cells (MSPCs) derived from adult rats in a spinal cord transection model. The level of axonal regeneration, the effect of chitosan channels on the survival of MSPCs, and the functional recovery results were also evaluated. MATERIAL AND METHODS: Chitosan channels stuffed with MSPCs were implanted at the level of T8 in a transected rat spinal cord. MSPCs were harvested from the pelvic bone marrow of adult rats, and the MSPC?chitosan channel group was compared with three control groups. The axonal regeneration capacity, the effect of chitosan channels on the survival of MSPCs, and the functional recovery results were compared among four groups. The survival of MSPCs was evaluated using histopathological techniques and electron microscopy, axonal regeneration/germination was evaluated by confocal microscopy, and locomotor function was assessed for 4 weeks using the Basso, Beattie, and Bresnahan locomotor score. RESULTS: The MSPC-chitosan channel group exhibited enhanced survival of transplanted MSPCs compared with MSPCs transplanted directly into the lesion cavity, although no significant difference was detected in locomotor function between the treatment and control groups. The MSPC-chitosan channel group demonstrated thicker myelination of axons than the other groups. CONCLUSION: Chitosan channels promoted the survival of transplanted MSPCs and created a tissue bridge after complete spinal cord transection. They also induced axonal regeneration and germination. No significant improvement in functional recovery was found between the groups.